This invention is useful in the pharmaceutical field, and, more detailedly, relates to novel indolopyrrolocarbazole derivatives inhibiting growth of tumor cells and exerting antitumor effect, and their use as antitumor agents.
Many compounds are already put to practical use as pharmaceuticals in the field of tumor chemotherapy. However, their effects are not always sufficient against various kinds of tumors, and the problem of resistance of tumor cells to these drugs also makes the methods of clinical use complicate (see, Proceedings of the 47th Annual Meeting of Japan Cancer Society, pages 12-15, 1988).
Under the circumstances, development of novel anticancer substances is always desired in the field of cancer treatment. Particularly, there is need for substances overcoming resistance to existing carcinostatic substances and showing effectiveness against cancers on which existing carcinostatic substances cannot exert sufficient effect.
Under these present circumstances, the present inventors have widely screened metabolites of microorganisms, and as a result, they found a novel compound BE-13793C (12,13-dihydro-1,11-dihydroxy-5H-indolo[2.3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione) having antitumor activity (see, EP-A2-0388956).
Thereafter, they have tried to create compounds having further excellent antitumor activity by chemically modifying BE-13793C, and disclosed such compounds in prior patent applications and patents (EP-A1-0528030, U.S. Pat. Nos. 5,591,842, 5,668,271 and 5,804,564, WO95/30682 and WO96/04293).
U.S. Pat. No. 5,668,271 discloses a wide range of compounds having antitumor activity, including compounds wherein R1 (or R2) is 1) a C1-C6 alkyl group substituted by a C6-C12 aromatic hydrocarbon group; or 2) a 5- or 6-membered nonaromatic or aromatic heterocyclic group. The compounds of the present invention have a xe2x80x94(CH2)m-R group (wherein R represents a substituted or unsubstituted group selected from pyridyl, furyl or thienyl) bound to the exocyclic nitrogen instead of R1 (or R2). Furthermore, as disclosed in the instance application, the compounds of the invention have much better antitumor effects than the analogous compounds generically and specifically disclosed in U.S. Pat. No. 5,668,271.
Although, in the structural formula shown in Example 35 of the above U.S. Pat. No. 5,668,271, a group corresponding to xe2x80x94(CH2)mxe2x80x94R in the compounds of the present invention is shown as a 2-pyridylmethyl group, this group should be read a 2-pyridylcarbonyl group. This is apparent from that xcex1-picolinohydrazide is used as a reactant.
Compounds disclosed in the above-mentioned other prior applications and patents are further remoter in structure from the compounds of the present invention. Namely, EP-A1-0528030 solely discloses compounds wherein a group correspoding to xe2x80x94NHxe2x80x94(CH2)mxe2x80x94R in the compounds of the present invention is H. U.S. Pat. No. 5,591,842 discloses compounds wherein groups corresponding to xe2x80x94(CH2)mxe2x80x94R and H bound to N in the compounds of the present invention are R1 and R2 and the R1 and R2 include comprehensive groups, but as compounds closest to the compounds of the present invention, only compounds wherein R1 and R2 are C1-C6 alkyl groups substituted with a C6-C12 aromatic hydrcarbonic group, or furyl, thienyl or pyridyl groups (one of R1 and R2 can be H) are disclosed. U.S. Pat. No. 5,804,564 and WO95/30682 solely disclose compounds wherein a group corresponding to xe2x80x94(CH2)mxe2x80x94R in the compounds of the present invention is a bis(hydroxymethyl)methyl group. WO96/04293 discloses compounds wherein a group corresponding to xe2x80x94NH(CH2)mxe2x80x94R in the compounds of the present invention is R1 and this R1 includes comprehensive groups, but compounds close to the compounds of the present invention are not found and, further, a group corresponding to G in the compounds of the present invention is a disaccharide group.
Some of the compounds of the present invention, namely compounds wherein R is a pyridyl, furyl or thienyl group each having a substituent are disclosed in JP-A-10-245390 (published on Sep. 14, 1998), the inventors of which are the applicants of the present application. The JP-A application has not yet been examined and applications to other countries corresponding thereto have not been made except the present application.
From the above, in relation with the above-mentioned background art, the invention of the present application are believed not to be considered at least as an object of rejection under 35 U.S.C. xc2xa7102 (B) and (D).
It is a problem to be solved in the present invention to create compounds having further excellent antitumor activity by chemically modifying indolopyrrolocarbazole antitumor substances disclosed in the prior patent applications.
For solving the above problem, the present inventors have synthesized a wide range of indolopyrrolocarbazole derivatives and examined their antitumor or activity, and found that compounds represented by the later-described formula [I] show further excellent antitumor activity than the indolopyrrolocarbazole compounds disclosed in the prior applications, and completed the present invention.
Namely, the invention relates to a compound represented by the formula or a pharmaceutically acceptable salt thereof 
wherein R represents an unsubstituted pyridyl, furyl or thienyl group, or a pyridyl, furyl or thienyl group each of which has one or more substituents selected from the group consisting of a hydroxyl group, a lower alkoxy group, a hydroxy lower alkyl group and a hydroxy lower alkenyl group except that when the pyridyl, furyl or thienyl group has a lower alkoxy group as a substituent, each of which simultaneously has another substituent selected from the group consisting of a hydroxyl group, a lower alkoxy group, a hydroxy lower alkyl group and a hydroxy lower alkenyl group, m represents an integer of 1 to 3, and G represents a xcex2-D-glucopyranosyl group, and the positions of substitution of the hydroxyl groups on the indolopyrrolocarbazole ring are the 1- and 11-positions, or the 2- and 10-positions, and a use thereof as an antitumor agent.
In the definition of the substituents in the compounds of the invention represented by the formula [I], lower alkoxy groups represent straight-chain or branched alkoxy groups having 1 to 6, preferably 1 to 4, further preferably 1 or 2, most preferably 1 carbon atoms, and there can for example be mentioned a methoxy group, an ethoxy group, a propoxy group, n-butoxy group, s-butoxy group, n-hexyl group, etc.
Hydroxy lower alkyl groups represent straight-chain or branched hydroxyalkyl groups having 1 to 6, preferably 1 to 4, further preferably 1 or 2, most preferably 1 carbon atoms, and there can for example be mentioned a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a hydroxyhexyl group, etc.
Hydroxy lower alkenyl groups represent straight-chain or branched hydroxyalkenyl groups having 2 to 6, preferably 3 or 4, further preferably 3 carbon atoms, and there can for example be mentioned a 3-hydroxy-1-propenyl group, etc. m represents an integer of 1 to 3, preferably 1.
The positions of substitution of the hydroxyl groups on the indolopyrrolocarbazole ring may be the 1- and 11-positions, or the 2- and 10-positions, but the 2- and 10-positions are preferred.
Description is made below on processes for preparing the compounds of the invention.
An indolopyrrolocarbazole derivative of the invention can be prepared by reacting a compound represented by the formula 
wherein, A represents NH or H, and G is as defined above, the compound being a known compound disclosed in EP-A1-0528030, EP-A1-0545195, WO95/30682 and WO96/04293, with a compound represented by the formula
H2Nxe2x80x94NH(CH2)mRxe2x80x83xe2x80x83[III]
wherein, R and m are as defined above, or by condensing a compound represented by the formula 
wherein, G is as defined above, with a compound represented by the formula
xe2x80x83R1(CH2)mCHOxe2x80x83xe2x80x83[V]
wherein, R1 has the same meaning as R or means R wherein hydroxyl groups are protected, and m is as defined above, then carrying out reduction, and, if necessary, removing the protective groups, or by reacting a compound of the formula [IV] with a compound represented by the formula
R1(CH2)mLxe2x80x83xe2x80x83[VI]
wherein, L means a leaving group, and R1 and m are as defined above, and, if necessary, removing the protective groups.
The reaction between the compound represented by the formula [II] and the compound represented by the formula [III] is reaction between an imide or an acid anhydride and a hydrazine derivative, well-known in the chemical field. This reaction can be carried out using a solvent usually having no bad influenece on the reaction, such as, for example, tetrahydrofuran or N,N-dimethylformamide. The use amount of the compound [III] is usually a little excess to 5 molar equivalents based on the compound [II], but a largely excess use of the former is possible.
The reaction temperature is usually in the range of xe2x88x9250xc2x0 C. to the boiling point of the solvent, but, if necessary, temperature higher than or lower than the temperature can be used. The reaction time is usually in the range of 30 minutes to 2 days, but time longer than or shorter than the time can be used.
The reaction of preparing a compound [I] by condensing a compound represented by the formula [IV] with a compound represented by the formula [V] and then carrying out reduction can be carried out in the same reaction system, but in some occasion, it is also possible to, once isolate the Schiff base as an intermediate product. Namely usually, the reaction can be carried out by mixing the compound [IV] with the compound [V] in a suitable solvent and then adding a reducing agent. The reaction is preferably carried out in the presence of an acid such as acetic acid or hydrochloric acid. As usable solvents, there can, for example, be mentioned alcoholic solvents such as methanol and ethanol, aprotic polar solvents such as N,N-dimethylformamide, etc. The reduction of the Schiff base can be carried out using a metal hydride complex such as sodium cyanoborohydride, or the like, and also by catalytic reduction.
The reaction between a compound [IV] and a compound [VI] is alkylation reaction of an amine, and can be carried out by a known method, for example by reaction with an alkyl halide, alkyl mesylate or alkyl tosylate or the like.
The products of the above reactions can be purified by methods known in the field of organic chemistry, for example by precipitation methods, solvent extraction methods, recrystallization, chromatography, etc.
Further in the invention, pharmaceutically acceptable salts of compounds obtained by the above processes are included. As such salts, there can be mentioned salts with an alkali metal such as for example potassium or sodium, salts with an alkaline earth metal such as for example calcium, salts with a basic organic compound such as for example ethylamine or arginine, salts with an inorganic acid such as hydrochloric acid or sulfuiric acid, and salts with an organic acid such as acetic acid, citric acid or maleic acid.
The compounds of the invention represented by the formula [I] show excellent antitumor action.
Growth Inhibition Activity on Mouse Leukemia Cells
100 xcexcl of a medium for cell culture (RPMI-1640 medium containing 10% fetal bovine serum) containing 3xc3x97103 mouse leukemia cells (P388) was put in a 96-well microplate, and culture was carried out at 37xc2x0 C. for 24 hours under 5% CO2. 10 xcexcl of a medium containing a test compound was added, and culture was continued at 37xc2x0 C. for further 24 hours under 5% CO2. 10 xcexcl of 0.5% Thiazoyl Blue was added to the cultured medium, and incubation was made at 37xc2x0 C. for 2 hours under 5% CO2 to carry out enzymatic reaction. 20% sodium dodecyl sulfate (SDS) was added to discontinue the reaction, and incubation was carried out at 37xc2x0 C. for further 4 hours to dissolve the resulting dye, and absorbance at 550 nm was measured and compared with the control group. The results are shown in Table 1.
Growth Inhibition Activity on Human Gastric Cancer Cells, Human Colon Cancer Cells and Human Lung Cancer Cells
100 xcexcl of a medium for cell culture (RPMI-1640 medium containing 10% fetal bovine serum) containing 1xc3x97104 human gastric cancer cells (MKN-45), human colon cancer cells (DLD-1) or human lung cancer cells (PC-13) was put in a 96-well microplate, and culture was carried out at 37xc2x0 C. for 24 hours under 5% CO2. 100 xcexcl of a medium containing a test compound was added, and culture was continued at 37xc2x0 C. for further 72 hours under 5% CO2. After completion of the culture, 50 xcexcl of 50% trichloroacetic acid was added to the cultured medium, and the mixtures were held at 4xc2x0 C. for 60 minutes to fix the cells. The cultured medium containing trichloroacetic acid was removed, and the wells were washed with tap water and dried. 50 xcexcl of 0.4% Sulforhodamine B solution was added, and the mixtures were held at room temperature for 30 minutes to dye the cells. The dyeing solutions were removed, and the remaining dyeing solutions were washed away with 1% acetic acid. After complete removal of the acetic acid solution and drying, 200 xcexcl of 10 nM Tris solution was added and the mixtures were mixed well at room temperature for 1 hour or more to extract the dye. Absorbance of each well at 560 nm was measured, and the inhibition proportion in comparison with the group of no addition of any drug was calculated. The results are shown in Table 1.
Control compound 1: a compound of the formula [I] of the present invention wherein xe2x80x94AAAAAAA(CH2)mxe2x80x94R is a 2-pyridyl group and the positions of substitution of the hydroxyl groups on the indolopyrrolocarbazole ring are the 1- and 11-positions. The control compound 1 is disclosed in Example 15 of U.S. Pat. No. 5,668,271.
Control compound 2: a compound of the formula [I] of the present invention wherein xe2x80x94(CH2)mxe2x80x94R is a 3-hydroxybenzyl group and the positions of substitution of the hydroxyl groups on the indolopyrrolocarbazole ring are the 1- and 11-positions. The control compound 1 is disclosed in Example 23 of U.S. Pat. No. 5,668,271.
Antitumor Effect on Human Gastric Cancer (MKN-45), Human Breast Cancer (MX-1) and Human Colon Cancer (LS180)
A MKN-45, MX-1 or LS180 solid tumor previously subcutaneously implanted in a nude mouse and grown was thinly cut, and its cubes with each side 3 mm were subcutaneously implanted in test mice. After the implantation, starting from the time when the tumor grew into 0.3 cm3 or more, various dosages of a test compound were injected once a day for 5 consecutive days into the tail veins of the mice. The same injections were then made for further 5 days after 2 days of interval (treatment schedule: 5/wxc3x972) or once every 3-4 days, four times in total (treatment schedule: 2/wxc3x972). 28 after the start of the treatment, the length (L) and the breadth (W) of each of the tumors were measured, and its volume (V) was calculated (V=xc2xdxc3x97Lxc3x97W2). A tumor growth inhibition proportion was calculated based on the volume, and a total dose to inhibit tumor growth by 75% (GID75, mg/m2 body surface area) was then determined. The results are shown in Table 2.
Control compound: a compound of the formula [I] of the present invention wherein xe2x80x94(CH2)mxe2x80x94R is a 3-hydroxybenzyl group and the positions of substitution of the hydroxyl groups on the indolopyrrolocarbazole ring are the 2- and 10-positions. This control compound is not specifically disclosed in U.S. Pat. No. 5,668,271, but is an isomer of the compound disclosed in Example 23 of the U.S. Patent, both compounds differing in the positions of substitution of the hydroxyl groups on the indolopyrrolocrbazole ring.
Compounds provided by the present invention show a much better antitumor action than the control compounds, as shown in the above pharmacological test results.
As apparent from the results of the above pharmacological tests, the compounds of the invention show an excellent antitumor action, and are useful as antitumor agents for humans or other mammals, particularly humans, for prophylaxis and/or treatment of, for example, head or neck cancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer, hepatic cancer, pancreatic cancer, colon cancer, renal cancer, prostate cancer, testoid cancer, uterine cancer, ovarian cancer, breast cancer, brain cancer, etc. as solid cancers, and leukemia, lymphoma, myeloma, etc. as other cancers, preferably gastric cancer, colon cancer, lung cancer and breast cancer.
A compound of the invention can be used in the form of antitumor pharmaceutical preparations suitable for oral administration, parenteral administration, etc., obtained by mixing the compound with solid or liquid excipients or carriers known in the field of pharmaceutical preparations. As forms for oral administration, there can be mentioned peroral agents such as for example tablets, capsuls, powders, granules and liquids, and as forms for parenteral administration, there can be mentioned sterilized liquid parenteral agents such as for example solutions and suspensions.
Solid preparations such as tablets, capsules, granules and powders can be prepared using compounds of the invention alone, but can also be prepared further using suitable additives. As the suitable additives, there can be mentioned conventional additives, for example, sugars such as for example lactose and glucose, starches such as for example corn, wheat and rice, fatty acids such as for example stearic acid, inorganic salts such as for example magnesium metasilicate aluminate and anhydrous calcium phosphate, synthetic macromolecules such as for example polyvinylpyrrolidone and polyalkylene glycols, fatty acid salts such as for example calcium stearate and magnesium stearate, alcohols such as for example stearyl alcohol and benzyl alcohol, synthetic cellulose derivatives such as for example methylcellulose, carboxymethylcellulose, ethylcellulose and hydroxypropylmethylcellulose, and further, water, gelatin, talc, vegetable oils, gum arabic, etc.
These solid preparations such as tablets, capsules, granules and powders can contain, generally 0.1 to 100% by weight, preferably 5 to 100% by weight of an effective ingredient.
Liquid preparations can be prepared as forms of suspensions, syrups, injections, etc. using suitable additives usually used in liquid preparations, such as water, alcohols or vegetable oils including soybean oil, peanut oil and sesame oil.
Particularly, as solvents suitable in parenteral administration including intramuscular injection, intravenous injection and subcutaneous injection, there can for example be mentioned distilled water for injection, aqueous lidocaine hydrochloride solution (for intramuscular injection), physiological saline, aqueous glucose solution, ethanol, polyethylene glycol, liquids for intravenous injection (e.g., aqeous solutions of cetric acid and sodium citrate, etc.), electrolyte solutions (for intravenous injection by drip and for intravenous injection), etc., or their mixed solutions.
These injections may include not only those wherein previous dissolution is made, but also those in the form of dissolving powder alone or with suitable additives when used. These injections usually contain 0.1 to 10% by weight, preferably 1 to 5% of an effective ingredient.
Liquid preparations such as suspensions and syrups for oral administration can contain 0.5 to 10% by weight of an effective ingredient.
It should be noted that the actually preferred dose of the compounds of the invention is varied depending on kinds of compounds used, kinds of compositions prepared, application frequency, particular sites to be treated, hosts and tumors. For example, the dose of each compound per day and per one adult is 10 to 500 mg in the case of oral administration, and 10 to 100 mg in the case of parenteral administration, preferably intravenous injection. The frequency of administration is varied depending on administration methods and symptoms, but the administration can be made in a time or in devided 2 to 5 times. Administration methods including intermittent administration such as every two-days administration or every three-days administration can also be used.